Monoclonal antibodies directed against the microtubule-associated protein tau, hybridomas secreting these antibodies, antigen recognition by these monoclonal antibodies and their applications

ABSTRACT

The invention relates to a monoclonal antibody which forms an immunological complex with an epitope of an antigen belonging to normal human tau protein as well as abnormally phosphorylated human tau protein, with said tau protein being liable to be obtained from a brain homogenate, itself isolated from human cerebral cortex. The monoclonal antibodies of the invention can be used to detect tau and abnormally phosphorylated tau in brain extracts and in unconcentrated cerebrospinal fluid.

PRIOR APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/403,917 filed Jan. 19, 1995, which is a continuation-in-part of U.S.patent application Ser. No. 08/403,916, filed Jan. 19, 1995, nowabandoned which is a continuation-in-part of U.S. patent applicationSer. No. 08/244,951, filed Jun. 13, 1994, which is acontinuation-in-part of PCT application Ser. No. 93003499, filed Dec.10, 1993.

The invention relates to new monoclonal antibodies directed against thehuman microtubule-associated protein tau, to the hybridomas secretingthese monoclonal antibodies, and to the antigen recognition by thesemonoclonal antibodies and their applications. The invention also relatesto a process for diagnosing brain diseases involving the particularepitope (of the tau protein) which is recognized by said monoclonalantibodies.

Alzheimer's disease (AD) is the most common form of adult-onsetdementia. At present, no biochemical test is available for antemortemdiagnosis of AD. The disease is therefore clinically diagnosed primarilyby exclusion of other forms of dementia. The illness is characterizedneuropathologically by the presence of neuritic (senile) plaques andneurofibrillary tangles (NFT).

Neurofibrillary tangles consist of paired helical filaments (PHF), ofwhich the main protein component is a modified form of themicrotubule-associated protein tau (Brion et al., 1985; Greenberg andDavies, 1990; Lee et al.,1991), which under normal circumstancespromotes microtubule assembly and stability (Weingarten et al., 1975;Bre and Karsenti, 1990), which is synthesized in the neurons of severalspecies, including humans (Kosik et al., 1989) and which is abundantlypresent in the axonal compartment of these neurons (Binder et al.,1985).

The protein exists as a family of different isoforms of which 4 to 6isoforms are found in normal adult brain but only 1 isoform is detectedin fetal brain (Goedert et al., 1989). The diversity of the isoforms isgenerated from a single gene by alternative mRNA splicing (Himmler,1989). The most striking feature of tau protein, as predicted frommolecular cloning, is a stretch of 31 or 32 amino acids occurring in thecarboxy-terminal part of the molecule that is repeated 3 or 4 times.Additional diversity is generated through 29 or 58 amino acid-longinsertions in the NH₂ -terminal part of the molecules (Goedert et al.,1989).

Tau variants of 64 and 69 kDa, which are abnormally phosphorylated, asrevealed by the (apparent increase in their molecular mass observedalter alkaline phosphatase treatment, have been detected exclusively inbrain areas showing neurofibrillary tangles and senile plaques (Flamentet al., 1989, 1990). The sites of phosphorylation by 4 different kinaseshave been mapped in the C-terminal microtubule-binding half of tau, andit could be shown that the action of a calcium calmodulin-dependentkinase on bacterially expressed tau resulted in the phosphorylation ofSer(405) which induced a lower electrophoretical mobility (Steiner etal., 1990). Tau present in paired helical filaments, called PHT-tau isabnormally phosphorylated (Lee et al., 1991). This abnormalphosphorylation causes a conformational change in tau, resultingprobably in self-association and the formation of PHFs. PHF-tau in AD isphosphorylated at several sites, one of which is the phosphoserine 199and/or 202. This site is specifically recognized by a mAb called AT8(Biernat et al., 1992). Therefore, AT8 is a discriminative marker forPHF-tau (Goedert et al., 1992).

Several antibodies have been reported that show reactivity to human taueither because they are directed to non-specific phosphorylated epitopespresent on neurofilament and subsequently shown to cross-react withnormal and abnormally phosphorylated tau (Nukina et al., 1987;Ksiezak-Reding et al., 1987) or because they recognized specificepitopes on normal and abnormally phosphorylated tau (Kosik et al.,1988). In addition to the tau antibodies directed towards non-specificepitopes, antibodies directed specifically to phosphorylated tauepitopes have been described (Mercken et al., 1992b).

Although overall tau mRNA levels are only slightly modulated inAlzheimer-affected brain regions (Goedlert et al., 1988; Barton et al.,1990) it has been shown that total tau protein levels may differ atleast 6-fold (Khatoon et al., 1992). This has been demonstrated bypolygonal antibodies against tau (Flament and Delacourte, 1990) and bymonoclonal antibodies directed to well-defined epitopes. The Alz 50monoclonal antibody recognizing a phosphate-independent epitope in theN-terminus of the tau molecule (Goedert et al., 1991) has been used in asandwich immunoassay on brain homogenates and it has been shown thattaul levels are higher in Alzheimer's patient brains (Ghanbari et al.,1990; patent application EP 444 856).

An antibody named "423", raised against pronase-treated PHFs andspecifically reactive with a 9.5 kDa and a 12 kDa fragment was also usedto measure tau protein in Alzheimer's disease (patent application WO89/03993). Similarly, it was found that increased mAb 423immnunoreactivity was observed in Alzheimer brain homogenates ascompared with control brain homogenates.

Mercken et al, (1992b) describe a range of monoclonal antibodies whichare either specific for a phosphatase-sensitive epitope (AT8) or whichreact with PHF-tau as well as with normal tau (AT1, AT4, AT6, AT9, AT11,AT12 and AT14) in Western blotting.

Moreover, the antibody tau 1 (Wischik et al., 1988; Harrington et al.,1990) was also used to measure tau in brain homogenates. In one casewhen tau levels were specifically measured in Alzheimer-affected brainsections, tau levels were eight-fold higher as compared with levels innormal brain homogenates (Khatoon et al., 1992).

In a first attempt to diagnose Alzheimer disease in cerebrospinal fluid,the PHF-tau-specific monoclonal antibody AT8 (Mercken et al., 1992b),was used. However, no PHF tau antigen could be demonstrated.

Thus far, none of the monoclonals that have been described have beensuccessful in detecting tau in non-concentrated cerebrospinal fluid(CSF), although the presence of tau was observed in 100-foldconcentrated CSF (Wolozin and Davies, 1987) or in CSF samples usingpolyclonal antibodies (Delacourte and Vermersch, 1991).

The aim of the present invention is therefore to provide monoclonalantibodies which allow reliable and sensitive detection of normal andabnormally phosphorylated tau present in brain extracts and inunconcentrated cerebrospinal fluid. The invention also aims at providingthe hybridoma which secretes the above-said monoclonal antibodies.

The invention furthermore aims at providing the epitope of the tauprotein present in brain homogenates or in body fluids such ascerebrospinal fluid, which is recognized by said monoclonal antibodies.

The invention aims at providing a process for the detection or diagnosisin vitro of brain diseases involving tau protein.

The monoclonal antibodies of the invention are characterized by the factthat they react with an epitope which is present in both normal andabnormally phosphorylated human tau protein. The monoclonal antibodiesare furthermore characterized by the fact that they form animmunological complex with an epitope or an antigen belonging to normaland abnormally phosphorylated human tau protein. The monoclonalantibodies of the invention are also characterized by the fact that theydo not form an immunological complex with other phosphorylated proteinssuch as MAP-1, MAP-2 and neurofilaments which share part of theirsequence with tau protein (Nukina et al., 1987; Lewis et al., 1988) asdetermined by means of an ELISA. The monoclonal antibodies of theinvention are also characterized by the fact that they are liable todetect human tau protein in CSF, with said tau protein being at aconcentration as low as 1.0 pg/ml and with said tau protein beingdetected at 100% recovery upon the addition of a fixed amount of tauprotein in tau protein-negative CSF (100% spiking recovery).

The monoclonal antibodies of the invention also enable the diagnosis ofAlzheimer's disease (AD) on the basis of CSF, i.e., to detect tau andmodified forms of tau in CSF. The problem associated herewith is thatthis antigen is present in a very low amount in CSF, therefore thedetection assay must be very sensitive. This problem can be resolved byusing the combination of the monoclonal antibody of the inventiontogether with the catalysed reporter deposition amplification technique(CARD, Bobrow et al., 1989), allowing a tau-specific CARD ELISA with ahigher sensitivity. Alternatively, a mixture or combinations of labeledmonoclonal antibodies, each recognizing epitopes different from AT120epitope, could be used as detector antibodies.

The results obtained with the monoclonal antibody secreted by thehybridoma AT120 of the invention indicate that elevated tau levels arenot only found in AD, but also in other neurological diseases whereneuronal death or damage occurs.

The expression "form an immunologically complex with" means that themonoclonal antibody of the invention binds to the above-said antigenunder one of the following conditions as mentioned in the techniquesbelow:

Light Immunomicroscopy

Brain tissue samples, obtained at surgery or autopsy, are fixed byimmersion in 4% formalin or Bouin's fixative and embedded in paraffinfor sectioning. The monoclonal antibodies of the invention are appliedin conjunction with a technique to visualize the formed immune complexessuch as the avidin-biotinylated peroxidase complex technique (Hsu etal., 1981) using 3,3'-diaminobenzidine tetrahydrochloride fordevelopment of color. Sections are counterstained with Harrishaematoxylin stain.

Immunoelectron Microscopy in Tissue Sections

Brain tissue samples, obtained at surgery or autopsy are fixed in eitherBouin's fixative or 10% buffered formalin before sectioning withoutembedding (Vibratome). The monoclonal antibody of the invention is usedfor immunostaining by the indirect immunogold method after which thesections are fixed, embedded and sectioned for electron microscopy, allaccording to standard protocols known to those skilled in the art (Brionet al., 1985).

Immunoblotting Procedures

For immunoblotting, fractions enriched in tau are prepared as described(Lindwall and Cole, 1984). Typically, 50 g of brain tissue is cut intosmall pieces with scissors and homogenized 1:1 (wt/vol) in buffer A (20mM 2- N-morpholino!ethanesulfonic acid, 80 mM NaCl, 2 mM EDTA, 0.1 mMEGTA, 1 mM β-mercaptoethanol, pH 6.75) with a Potter homogenizerequipped with a Teflon plunger. The homogenate is centrifuged for 1 h at150,000 g at 4° C., and the supernatant is heated for 5 min in boilingwater and chilled again for 10 min on ice. The slurry is centrifuged for2 h at 150,000 g at 4° C., and the supernatant is collected. Theheat-stable cytosolic extract is added to 2.5% perchloric acid andcentrifuged for 1at 150,000 g at 4° C., after which the supernatant isneutralized with 3M Tris. The supernatant is then dialyzed in water andconcentrated in a Centriprep concentrator (Amicon, Lausanne,Switzerland).

SDS-polyacrylamide electrophoresis is performed under reducingconditions on 12% gels (Laemmli, 1970). After electrophoresis, theproteins are either fixed and stained with Coomnassie brilliant blue, ortransferred (Towbin et al., 1979) to nitrocellulose sheets (Hybond-C,Amersham) or Immobilon filters (Millipore).

After transfer, the filters are presoaked in PBS containing 0.05% (v/v)Tween 20 (Tween-PBS) and then incubated for 1 h in Tween-PBS containing5% (w/v) skimmed dried milk and 10% (v/v) newborn calf serum (blockingbuffer). Next, the filters are treated overnight at 4° C. with amonoclonal antibody according to the invention appropriately diluted inblocking buffer.

The filters are then washed three times in Tween-PBS and treated for 1.5h at room temperature with horseradish peroxidase-labeled rabbitanti-mouse IgG (Dakopatts, Denmark) diluted 1/3000 in blocking buffer.After three washes in Tween-PBS, streptavidine-biotinylated horseradishperoxidase complex (Amersham), diluted 1/250 in blocking buffer, isapplied for 1.5 h at room temperature. Thereafter, the filters arewashed three times in Tween-PBS and once in PBS. The filters are thenincubated in PBS containing 0.05% (w/v) diaminobenzidine and 0.03% (v/v)hydrogen peroxide until background staining develops.

It should be clear that the formation of an immunological complexbetween the monoclonal antibodies and the antigen is not limited to theprecise conditions described above, but that all techniques that respectthe immunochemical properties of the antibody and antigen binding willproduce similar formation of an immunological complex.

Human normal tau is a class of at least six tau proteins ranging inmolecular weight from 58 to 64 kDa which are specifically expressed inthe somatodendritic domain of all neurons (Papasozomenos and Binder,1987). Moreover Alzheimer (tangle)-specific tau forms have beendescribed which occur in the degenerating cortical neurons ofAlzheimer's disease or Down's Syndrome and of which the lowerelectrophoretic mobility can be attributed to abnormal phosphorylation(Flament et al., 1989; Delacourte et al., 1990).

According to an advantageous embodiment of the invention, the monoclonalantibody forms an immunological complex with all forms of tau describedabove, with said human tau protein being liable to be obtained from abrain homogenate, itself isolated from the cerebral cortex of a patientsuffering from a neurological disease.

A "brain homogenate" and tau protein can be obtained by the man skilledin the art according to standard methods such as the method of Lindwalland Cole (1984).

According to an advantageous embodiment, the monoclonal antibodies ofthe invention form an immunological complex:

either with an epitope located within the following amino acid sequenceof human tau protein: ##STR1## more specifically with an epitope locatedwithin the following amino acid sequence: ##STR2## more specificallywith an epitope located within the following amino acid sequence:##STR3## most specifically with an epitope located within the followingamino acid sequence: ##STR4## or with any other peptide capable offorming an immunological complex with a monoclonal antibody, which iscapable of forming a complex with an epitope located in a tau proteinregions as shown in any of SEQ ID NO 1 to 4.

The sequences as shown in SEQ ID NO 1 to 4 will be hereafter designatedas containing "the epitope" of the invention. Amino acid sequence 1spans the amino acids 155-221 of human tau using the numbering of humantau 40, amino acid sequence 2 spans the amino acids 199-231 of humantau, amino acid sequence 3 spans the amino acids 207-219 of human tau,and amino acid sequence 4 spans the amino acids 218-224 of human tau(Goedert et al., 1989).

The peptides capable of forming an immunological complex with amonoclonal antibody, which itself is liable to form a complex with theabove-mentioned peptide will be defined as the "variant peptides".

A preferred monoclonal antibody of the invention is secreted by thehybridoma deposited at ECACC (European Collection of Animal CellCultures, Vaccine Research and Production Laboratory, Public Health andLaboratory Service (PHLS), Centre for Applied Microbiology and Research,Proton Down, GB-Salisbury, Wiltshire SP4 OJG) on Oct. 8, 1992 under No.92100853.

This hybridoma will be hereafter designated as "hybridoma AT120"and thesecreted monoclonal antibody will be designated as "monoclonal antibodyAT120".

The invention also relates to the hybridoma which secretes a monoclonalantibody according to the invention, and particularly the hybridomafiled at ECACC on Oct. 8, 1992 under No. 92100853.

The above-mentioned monoclonal antibodies are obtained by a processinvolving obtention and isolation of hybridomas which secrete thesemonoclonal antibodies.

A process for obtaining the hybridoma involves:

starting from spleen cells of an animal, e.g. mouse or rat, previouslyimmunized in vivo, or from spleen cells of such cells previouslyimmunized in vitro with an antigen recognized by the monoclonal antibodydeposited at ECACC on Oct. 8, 1992 under No. 92100853, or with a peptidecontaining or which are constituted by parts of any of the sequences asrepresented in SEQ ID NO 1 to 4;

fusing said immunized cells with myeloma cells under hybridoma-formingconditions; and

selecting those hybridomas which secrete the monoclonal antibodies whichspecifically recognize an epitope of the above-said antigen and whichform an immunological complex with normal tau or the abnormallyphosphorylated form of tau protein or with the peptide comprising theepitope of tau recognized by the monoclonal antibody of the invention.

A process for producing the corresponding monoclonal antibodiesinvolves:

culturing the selected hybridoma as indicated above in an appropriateculture medium; and

recovering the monoclonal antibodies secreted by said selectedhybridoma; or alternatively

implanting the selected hybridoma into the peritoneum of a mouse and,when ascites has been produced in the animal;

recovering the monoclonal antibodies then formed from said ascites.

The monoclonal antibodies of the invention can be prepared byconventional in vitro techniques such as the culturing of immobilizedcells using e.g. hollow fibers or microcapsules or such as the culturingof cells in homogeneous suspension using e.g. airlift reactors orstirred bioreactors.

The invention also relates to a peptide (antigen), which can be obtainedfrom a human brain homogenate itself being isolated from the humancerebral cortex obtained from a patient having Alzheimer's disease, andwhich forms an immunological complex with the monoclonal antibody of theinvention.

The invention also relates to peptides (antigens) which are liable toform an immunological complex with anyone of the monoclonal antibodiesof the invention and

which are contained in or are constituted by parts of the sequence asshown in SEQ ID NO 1, 2, 3, or 4;

which contain or are constituted by the sequence of the variant peptidesdefined above.

It is to be recalled that variant peptides are those peptides able toform an immunological complex with a monoclonal antibody, which itselfis liable to form a complex with an epitope located in the tau proteinregion as shown in SEQ ID NO 1 to 4.

The invention also relates to polypeptides (antigens) of about 100 aminoacids

which contain the sequence as shown in SEQ ID NO 1 to 4, or

which contain the sequence of the variant peptides defined above.

The invention also relates to the above-mentioned peptides which areliable to generate monoclonal antibodies of the invention.

The invention also relates to a peptide (antigen) which is contained inthe brain, in the cerebrospinal fluid, or in the serum of a patienthaving Alzheimer's disease or any brain disease involving normal humantau protein and which forms an immunological complex with a monoclonalantibody of the invention.

The invention also relates to a peptide (antigen) which is contained inthe brain, in the cerebrospinal fluid, or in the serum of a patienthaving Alzheimer's disease or any brain disease involving PHF orabnormally phosphorylated human tau protein and which forms animmunological complex with a monoclonal antibody of the invention.

A method for preparing the peptides of the invention is characterized inthat, preferably starting from the C-terminal amino acid, the successiveaminoacyl in the requisite orders or aminoacyls and fragments formedbeforehand and already containing several aminoacyl residues in theappropriate order, or alternatively several fragments prepared in thismanner beforehand, are coupled successively in pairs, it beingunderstood that care will be taken to protect all the reactive groupscarried by these aminoacyls or fragments beforehand except for the aminegroups of one and carboxyl groups of the other, or vice versa, whichmust normally participate in peptide bond formation, in particular afteractivation of the carboxyl group, according to methods known in peptidesynthesis, and so on, proceeding stepwise up to the N-terminal aminoacid.

The antigen of the invention, which can be prepared by methods known tothose skilled in the art (Lindwall and Cole, 1984) starting from thehuman cerebral cortex is characterized by its ability to form animmunological complex with the monoclonal antibody of the invention asdefined above, advantageously with the monoclonal antibody secreted bythe hybridoma AT120 deposited at the ECACC under No. 92100853 on Oct. 8,1992.

The antigen of the invention is advantageously contained in the brain,in the cerebrospinal fluid or the serum of a patient having Alzheimer'sdisease, Down syndrome, Pick's disease, subacute sclerosingpanencephalitis (SSPE) or other neurological diseases in which thenormal tau or abnormally phosphorylated tau protein are implicated; thisantigen provokes an immunological reaction with the monoclonal antibodyof the invention.

The invention also relates to a process for the detection or thediagnosis in vitro of brain disease involving tau protein, i.e.Alzheimer's disease, which involves:

bringing the monoclonal antibody of the invention into contact with apreparation of NFT containing tau protein or a detergent-extracted brainhomogenate containing tau protein isolated from a patient having hadAlzheimer's disease or any other disease involving tau protein orabnormally phosphorylated tau protein under conditions suitable forproducing an antigen-antibody complex; and,

separating the antigen from said complex and recovering the antigensought in a purified form.

The preparation of tau can be carried out according to Lindwall and Cole(1984).

Advantageously, the monoclonal antibodies used are in an immobilizedstate on a suitable support such as a resin. The process for thedetection of the antigen can then be carried out as follows:

bringing the supernatant containing proteins and polypeptides obtainedas a result of an extraction procedure starting from brain tissues orcerebrospinal fluid known to those skilled in the art (Iqbal et al.,1984; Greenberg and Davies, 1990) into contact with said monoclonalantibody, under such conditions as to allow the formation of animmunological complex;

washing the immobilized antibody-antigen complex then formed;

treating this complex with a solution (e.g. 3M potassium thiocyanate,2.5M magnesium chloride, 0.2M citrate-citric acid, pH 3.5 or 0.1M aceticacid) capable of producing the dissociation of the antigen-antibodycomplex; and;

recovering the antigen in a purified form.

The process of the invention for the detection or diagnosis in vitro ofbrain disease involving tau protein and abnormally phosphorylated tauprotein, as e.g. in Alzheimer's disease, includes:

bringing a sample of a brain homogenate, or of cerebrospinal fluid, orof serum from a patient suspected of suffering from a neurologicaldisorder involving tau protein or PHF, more particularly Alzheimer'sdisease, into contact under in vitro conditions with the monoclonalantibody of the invention, with said conditions being suitable forproducing an antigen-antibody complex; and

detecting the immunological binding of said antibody to said sample ofbrain homogeniate, or of cerebrospinal fluid, or of serum.

The detection of the immunologically bound monoclonal antibody can beachieved by conventional technology. Advantageously, the monoclonalantibody of the invention itself carries a marker or a group for director indirect coupling with a marker as exemplified hereafter. Also, apolyclonal antiserum can be used which was raised by injecting theantigen of the invention in an animal, preferably a rabbit, andrecovering the antiserum by immunoaffinity purification in which saidpolyclonal antibody is passed over a column to which said antigen isbound and eluting said polyclonal antibodies by conventional technology.

Detection can also be achieved by competition binding of the antigenwith a labeled peptide comprising the epitope of the invention.

A particularly advantageous embodiment of the process of the inventionfor the detection or diagnosis in vitro of brain diseases involving PHFand/or normal tau protein, e.g. Alzheimer's disease, comprises the stepsof:

bringing a sample of unconcentrated cerebrospinal fluid sample isolatedfrom a patient suspected of suffering from a neurological disorderinvolving normal or abnormally phosphorylated tau protein, moreparticulary Alzheimer's disease, into contact under in vitro conditionswith a monoclonal antibody according to the invention, under conditionssuitable for producing an antigen-antibody complex; and,

detecting the immunological binding of said antibody to said sample ofcerebrospinal fluid by means of a sandwich ELISA, preferably by applyingthe catalysed reporter diagnosis enhancement (CARD) procedure.

The invention also relates to a kit for the diagnosis in vitro of one ofthe following diseases: Alzheimer's disease, Down's syndrome, Pick'sdisease, subacute sclerosing panencephalitis (SSPE) and otherneurodegenerative disorders in which normal tau protein or abnormallyphosphorylated tau protein are implicated. Such a kit would contain:

at least one microplate for deposition thereon of any monoclonalantibody of the invention;

a preparation containing the sample to be diagnosed in vitro, possiblytogether with a labeled peptide containing the epitope of the inventionand preferably, a peptide lying in the peptide sequence as shown in SEQID NO 1 to 4,

a second antibody

which can be a monoclonal antibody recognizing another epitope of normalor abnormally phosphorylated tau protein, or of any peptide of theinvention, with said epitope being difterent from the one of theinvention, or

which can be a polyclonal antibody directed against normal or abnormallyphosphorylated tau, or against a peptide of the invention, with saidpolyclonal antibody being liable to form an immunological complex withepitopes which are all different from the epitope of the invention, withsaid polyclonal antibody being preferably purified by immunoaffinitychromatography using immobilized tau protein, or

a marker either for specific tagging or coupling with said secondantibody;

appropriate buffer solutions for carrying out the immunological reactionbetween the monoclonal antibody of the invention and a test sample onthe one hand, and the bound second antibody and the marker on the otherhand.

The labeled peptide mentioned above can be a peptide which has beenlabeled by any means known for the man skilled in the art. Moreover, themarker specific for tagging and coupling can be any marker known to theman skilled in the art.

The invention also relates to a kit, as described above, also containingthe antigen of the invention, with said antigen of the invention beingeither a standard (for quantitative determination of the antigen whichis sought) or a competitor, with respect to the antigen which is sought,for the kit to be used in a competition dosage process.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Western blotting detection of normal tau or PHF-tau using the monoclonalantibodies Tau-1, (Binder et al., 1985) AT8 (Mercken et al., 1992b) andAT120. Lanes 1, 3 and 5: PHF-tau isolated according to Greenberg andDavies (1990). Lanes 2, 4 and 6: Normal affinity purified human tauaccording to Mercken et al. (1992a). Lane 7: Molecular weight markers.

Lanes 1 and 2 were developed using AT8, lanes 3 and 4, using AT120 andlanes 5 and 6, using Tau-1 monoclonal antibody.

FIG. 2A and FIG. 2B

Detection of tau protein by immunochemistry

FIG. 2A: section from hippocampus of a patient with Alzheimer's disease.Magnification 212×.

FIG. 2B: section from another hippocampus of a patient with Alzheimer'sdisease with abundant tangles. Magnification 212×.

FIG. 3

Titration of normal (0) and PHF tau (▪), spiked in a tau-negative CSFpool using the amplified (CARD) AT120 sandwich ELISA. All dilutions weretested in duplicate and the data presented as optical density (OD)units.

FIG. 4

Western blotting of several deletion mutants constructed as indicated inexample V, and stained with AT120 as indicated in Example 1. Mutantscomprise the following amino acids (AA); Lane 1, full length tau 34(Goedert et al., 1989); Lane 2, amino terminus of tau 34 up to AA₁₅₄ ;Lane 3, from AA₁₅₅, to the carboxyterminus of tau 34; Lane 4;aminoterminus of tau 34 up to AA₂₄₂ ; Lane 5, aminoterminus tau 34 up toAA₂₂₂,; Lane 6, from AA₂₂₂ to the carboxyterminus of tau 34.

FIG. 5

Epitope recognition sites of the monoclonal antibodies HT7, BT2, AT8 aredepicted on the epitope of the invention (SEQ ID NO 1) shown in the oneletter amino acid code. Epitopes are boxed. The star "*" designates thefact that the AT8 epitope recognition needs phosphorylation of serineresidue 202.

FIG. 6A and 6B

Complete sequence of the mTHFMPH-tau 1 fusion protein (SEQ ID NO 23)with indication of relevant restriction sites on the nucleotide sequence(SEQ ID NO:25).

EXAMPLES Example I

Preparation of the monoclonal antibody AT120 (IgG1, subtype kappa)

1. Preparation of the antigen for immunization

PHF-tau was partially purified by a modification of the method ofGreenberg and Davies (1990). Postmortem tissue, consisting mostly ofgray matter from the frontal and temporal cortex, was obtained fromhistologically confirmed Alzheimer patients. This Alzheimer gray matterbrain sample (5-10 g) was homogenized with 10 volumes of cold buffer H(10 mM Tris/1 mM EGTA/0.8M NaCl/10% sucrose, pH 7.4) in a Teflon/glassPotter S (Braun, Germany) homogenizer. After centrifugation of thehomogenate in a 60 Ti MSE rotor at 27,000×g for 20 min at 4° C., thepellet was removed and the supernatant was adjusted to 1% (wt/vol)N-laurosylsarcosine and 1% (vol/vol) 2-mercaptoethanol and incubatedwhile rotating on a mixer (Swelab, Sweden) for 2.5 hours at 37° C. Thesupernatant mixture was centrifuged at 108,000×g for 35 min at 20° C.The PHF-tau containing pellet was gently washed with PBS and finallysuspended in 1 ml of the same buffer.

The antigen preparation was evaluated by a 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by , Westernblotting using immunoblotting with polyclonal rabbit antihuman normaltau antiserum (Mercken et al., 1992b).

2. Immunization protocol and fusion procedure

Balb/c mice were primed subcutaneously with 100 μg partially purifiedPHF-tau in complete Freund's adjuvant and boosted intraperitoneally 3times thereafter at 3-week intervals with 100 μg of the same antigen inincomplete Freund's adjuvant. On days 3 and 2 before the fusion, micewere boosted with 100 μg PHF-tau in saline.

Mouse spleen cells were fused with SP2/0 myeloma cells, using a modifiedprocedure of Kohler and Milstein (1975), with PEG 4000.

The cells of the fusion experiment were suspended at a density of4.5×10⁴ spleen cells/well on 96-well plates preseeded with mouseperitoneal macrophage cells as a feeder layer. These wells were screenedafter 12 days for anti-tau antibody production in a sandwich ELISAeither specific for normal tau or for PHF-tau as discussed in section 3.below.

Hybridoma growth was in Dulbecco's modified Eagle's medium (DMEM)supplemented with 20% fetal calf serum, sodium pyruvate (1 mM),L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), andnonessential amino acids. All products were purchased from Gibco,(Paisley, U.K.). Cells were incubated in a humidified CO₂ -airincubator.

3. Sandwich ELISA for antibody screening

The screening ELISA used for the detection of anti-tau monoclonalantibodies was a sandwich ELISA system with affinity-purified polyclonalrabbit anti-human tau antibodies (Mercken et al; 1992a) in the coatingphase. To this end, purified human normal tau, prepared as described inMercken et al. (1992a) was used for the preparation of an immunoaffinitycolumn using cyanogen bromide-activated Sepharose (Pharmacia, LKBSweden). The affinity-bound anti-tau fraction was eluted from thiscolumn with a 0.1M citric acid buffered solution at pH 2.5. Afterneutralization, the anti-tau-containing fractions were pooled and coatedovernight (1 μg/ml) at 4° C. on high-binding microtiter plates (Nunc,Gibco, Paisley, UK) in coating buffer (10 mM Tris, 10 mM NaCl, 10 mMNaN₃, pH 8.5). After overcoating for 30 min with 125 μl 0%-saturatedcasein in PBS to reduce non-specific binding, the plates were incubatedwith 100 μl of an appropriately diluted PHF-tau preparation andincubated for 60 min at 37° C. The plates were washed 3 times withPBS-0.05% Tween 20 (v/v); 100 μl hybridoma supernatant was added andincubation was continued for 1 h at 37° C. After washing, the boundmonoclonal antibodies were detected with peroxidase-conjugated rabbitanti-mouse serum (Dakopatts, Glostrup, Denmark). All reagents werediluted in PBS with 10% casein. After final washing, 100 μl 0.42 mM3,5,3',5'-tetramethylbenzidine, 0.003% H₂ O₂ v/v in 100 mM citric acid,100 mM disodium hydrogen phosphate, pH 4.3, was added as peroxidasesubstrate. The reaction was stopped with 50 μl of a 2M H₂ SO₄ solution.Absorbance was read in a Titertek Multiscan (Flow Laboratories, Eflab,Oy, Finland) at 450 nm.

The cross-reactivity of the monoclonal antibodies with normal tau inELISA was tested in a sandwich ELISA identical to the screening assay,except that affinity-purified normal tau was used as the antigen insteadof PHF-tau.

At the first selection of positive hybridoma cultures, most positivecultures were initially composed of mixed clones as seen by visualinspection of the wells (3-4 clones per well). These positive cultureswere arbitrarily designated AT1 to AT24 (some of these hybridomacultures, i.e., AT1 to AT14 were described by Mercken et al, 1992b).After this initial screening round, hybridoma cultures were subcloned bylimiting dilution, a technique well-know to those skilled in the art,finally resulting in pure hybridoma clones secreting antibodies with ahomogeneous idiotype. These pure hybridoma clones were further testedwith respect to their reactivity pattern on normal and PHF-tau in ELISA,Western blotting and immunohistochemistry, and to their capability todiagnose neurological diseases by means of their affinity for tauprotein present in an undiluted sample of cerebrospinal fluid. Based onthese criteria, the monoclonal antibody AT120 was selected and furthercharacterized as shown in the following examples.

4. Determination of the antibody class and subclass

The antibody class and subclass was determined by Inno-LIA(Innogenetics, Ghent, Belgium). The antibody of the invention, AT120,appeared to be of the IgG1, kappa subtype.

Example II

Detection of pathological tau and normal tau in ELISA and by Westernblotting

1. Detection of normal tau in ELISA using AT120.

Protein G-purified monoclonal antibody AT120, obtained from serum-freehybridoma AT120 conditioned medium, was coated on ELISA plates andreacted with different dilutions of affinity-purified human normal tauas described in Mercken et al. (1992a), prepared in a solution of PBSand 10% casein.

The purity of normal tau was determined by SDS-PAGE. Tau samples wereanalyzed on an 420 A/H amino acid analyzer (Applied Biosystems B.V.,Maarssen, The Netherlands) according to the manufacturer's instructionsand the protein showed the expected amino acid composition. From theamino acid composition and by comparison with a standard peptide theconcentration of normal tau was determined.

After incubation of the ELISA plates with different concentrations oftau spiked in tau- and PHF-tau-negative CSF for 1 h at room temperature,the plates were washed and incubated with 0,2 μg/ml biotynilated BT2 andHT7, each recognizing an epitope different from the AT120 epitope andpresent on normal term. After washing, complexed biotynilated antibodieswere detected with horseradish peroxidase conjugated streptavidine(Jackson) and color development as specified in example I. The resultsare shown in Table I and FIG. 3.

                  TABLE 1    ______________________________________    Detection of normal tau in ELISA                      ABSORBANCE                      (expressed as milli-    CONCENTRATION     absorbance units)    (pg/ml)           PHF-tau normal tau    ______________________________________    160               1682    1609    80                901     970    40                566     678    20                257     256    10                143     154     0                 92      87    ______________________________________

2. Detection of pathological tau and of normal tau in Western blottingusing AT120.

Purified normal human tau and PHF-tau were applied to 10%SDS-polyacrylamide gels and run under denaturing conditions according toLaemmli (1970). After SDS-PAGE, the transfer to nitrocellulose(Hybond-C, Amersham, Brussels, Belgium) was carried out in 10 mM NaHCO₃,3 mM Na₂ CO₃, pH 9.9 for 120 min at 55V with cooling. After blotting,the nitrocellulose was equilibrated to phosphate buffered saline (PBS),and protein binding sites were blocked with blot buffer (PBSsupplemented with 5% w/v skimmed dried milk and 10% v/v newborn calfserum). Blotted proteins were incubated overnight at 4° C. with AT120 asprimary antibody. After 3 washings with PBS-0.05% Tween 20(v/v),horseradish peroxidase-labeled rabbit anti-mouse immunoglobulins(Dakopatts, Glostrup, Denmark) were used at a dilution of 1/3000 andwere incubated for 90 min at room temperature. All antisera were dilutedin blot buffer. The blots were then washed three times in PBS/Tween anddeveloped with substrate solution (PBS,0.05% w/v 3,3'-diaminobenzidine,0.03% v/v H₂ O₂) after which the reaction was stopped in H₂ O. Resultsshown in FIG. 1 indicate, that the AT120 antibody recognizes all tauisoforms. By contrast, the Tau-1 antibody (Binder et al., 1985) reactssolely with normal tau, and the AT8 antibody (Mercken et al., 1992b)only with PHF-tau.

AT120, AT8 and Tau-1 mAbs were tested for phosphatase sensitivity oftheir epitopes in ELISA and in Western Blot on PHF-tau antigen. Thereactivity of the AT120 antibody with PHF-tau was not sensitive tophosphatase treatment either in ELISA (data not shown) or on Westernblots (data not shown). The reactivity of AT8 was almost completelyabolished after alkaline phosphatase treatment of the PHF-tau antigen inELISA. Dephosphorylation of PHF-tau enhanced Tau-1 immunoreactivity, asdescribed previously (Binder et al., 1985).

Example III

Detection of tau by immunohistochemistry

Paraffin sections of formalin-fixed brain tissue from neocortex,hippocampus, cerebellum, pons, and spinal cord of several Alzheimerpatients and age-matched controls were prepared, as well as sections ofperipheral nerve from one control patient.

Cryostat sections from Alzheimer and age-matched control brain were alsoprepared. Tissues were immunostained either with theperoxidase-antiperoxidase (PAP) technique (Steinberger et al., 1970) orwith the avidin-biotin complex (ABC) technique (Hsu et al., 1981) usingDakopatts (Denmark) and Amersham (UK) reagents, respectively.

Briefly, after blocking non-specific interactions with normal swineserum (Dakopatts X901) diluted 1:25 in Tris-buffered saline (TBS)containing 1% bovine serum albumin (BSA), sections were incubatedovernight with the AT120 primary antibody appropriately diluted inTBS/BSA. Secondary antibody and peroxidase complex were then applied for30 min each, with intermediate rinsing in TBS. Color was developed with3,3'-diaminobenzidinetetrahydrochloride (Sigma). Sections werecounterstained with Harris' hematoxylin, dehydrated, coverslipped, andviewed under a light microscope.

FIGS. 2A and 2B clearly indicate that AT120 produces abundant stainingof NFT, dystrophic neurites in plaques, and dispersed staining ofneuropil (neuropil threads).

Example IV

Detection of tau in cerebrospinal fluid samples

Cerebrospinal fluid samples

CSF samples from patients were collected at the Department of Neurologyof the University Hospital of Antwerp. All samples were obtained bylumbar puncture performed for routine diagnostic purposes. CSF sampleswere frozen and kept at -75° C. in small aliquots until use.

The patients were divided into 3 different groups: 27 patients diagnosedwith probable AD according to Mc Khann et al. (1984), mentally healthycontrol patients, who underwent lumbar puncture for radiculopathy andpatients suffering from other neurological diseases (OND). The OND groupincluded inflammatory, vascular, and other diseases, including patientswith neurodegenerative diseases such as adenoleukodystrophy, frontallobe degeneration, cerebellar atrophy, olivo-ponto-cerebellar atrophy,and amyotrophic lateral scelerosis. The age, sex and diagnosis werenoted for each patient.

AT120 assay

AT120 monoclonal antibodies purified from serum-free conditioned mediumby Protein G column chromatography were coated overnight at 4° C. onhigh-binding microtiter plates (Nunc, Gibco, Paisley, UK) in coatingbuffer at 3 μg/ml (10 mM Tris, 10 mM NaCl, 10 mM NaN₃, pH 8.5). Afterovercoating for 30 min with 150 μl 10%-saturated casein in PBS to reducenon-specific binding, the plates were incubated with 25 μl CSF and 75 μlconjugate mixture containing 0.2 μg/ml of biotynilated BT₂ and an equalamount of HT7 in 5% Tween 20, 10% saturated casein in PBS. The plateswere left overnight at room temperature and after washing peroxidaseconjugated streptavidine (Jackson) (1/15000) was added for 30 minutes atroom temperature.

Following an additional washing, 100 μl 0.42 mM 3.5, 3',5'-tetramethylbenzidine 0.003% H₂ O₂ v/v in 100 mM citric acid, 100 mMdisodium hydrogen phosphate, pH 4.3, were added as perocidase substrate.The reaction was stopped with 50 μl of a 2M H₂ SO₄ solution. Absorbancewas read in a Titertech Multiscan (Flow laboratories, Eflab, Oy,Finland) at 450 nm,

Absorbance values obtained with AT120 from the CSF samples were comparedwith standard curves generated from known quantities of affinitypurified normal human tau and this comparison allowed the result to beexpressed as pg tau/ml.

A summary of these results are compiled in Table II, where patients ID,diagnosis, age and tau values expressed in pg/ml CSF are listed. Fromthese results, it is obvious that levels of control patients aresubstantially lower (mean: 16.4 pg/ml) as compared to the group ofpatients suffering from various neurological diseases (OND; mean value:26.4 pg/ml). For patients with Alzheimer's disease the mean value isclearly elevated above those of control and OND samples (mean Alzheimerpatient: 50.8 pg/ml). If a cut-off level of 27 pg/ml is adopted, 8% ofthe control samples are positive, while for the OND group and theAlzheimer group these values are 27% and 80% respectively.

                  TABLE II    ______________________________________    Tau levels as determined with the AT120 ELISA assay, grouped according    to control patients, Alzheimer patients (AD) and other neurological    diseases (OND) and according to age cohort, expressed in pg/ml. Mean    and Std (=SD) is expressed as pg/ml per age cohort.    Number          Diagnosis         Age    pg/ml mean Std    ______________________________________    3     AD (early onset AD)                            35     * 56.5                                         33.3 12.3    260   A.D.              41     * 31.2    113   A.D.              44     * 42.6    161   A.D. (possibly Creutzfeld)                            57     * 33.8    81    A.D.              58     * 14.5    126   A.D.              59     * 24.8    421   A.D.              59     * 29.8    338   A.D.              64     * 51.2                                         61.68                                              35.4    254   A.D.              66     * 80.2    209   A.D.              67     * 74.4    383   Primary degenerative                            67     * 32.5          dementia    38    A.D.              67     * 68.7    229   A.D.              73     * 70.9    132   A.D.              76     * 51.9    88    A.D.              76     * 25.3    65    Dementia          77     * 80.1    71    A.D.              78     * 53.9    28    A.D. (early onset A.D.)                            78     * 48.7    11    A.D.              85     * 14    39    A.D.              86     * 150    386   control           5      * 17.5                                         17.3 4.37    108   control           20     * 14.3    402   control           26     * 14    106   control           27     * 14    424   control           28     * 14    355   control           28     * 20.1    399   control           29     * 14    373   control           30     * 17.4    381   control           31     * 14    372   control           32     * 19.2    379   control           32     * 18.3    241   control           32     * 14    415   control           32     * 14    428   control           33     * 16.6    118   control           34     * 21.5    224   control           36     * 15.7    24    control           37     * 25.2    369   control           38     * 23.9    425   control           39     * 14    145   control           40     * 31.9    61    control           40     * 17.3    377   control           40     * 14    366   control           41     * 17.7    401   control           41     * 20.1    400   control           42     * 14    417   control           42     * 14    354   control           43     * 16.8    34    control           43     * 25.8    217   control           43     * 14    364   control           44     * 14    134   control           45     * 16.7    367   control           45     * 14    427   control           45     * 14    394   control           48     * 14    361   control           48     * 14    237   control           50     * 17.2    396   control           52     * 16.4    100   control           54     * 23.9    411   control           54     * 14    371   control           55     * 18.4    423   control           56     * 14.9    99    control           56     * 27.9    192   control           60     * 19.9                                         24.83                                              15.17    387   control           61     * 14    389   control           65     * 19.5    426   control           66     * 15.4    141   control           67     * 14    368   control           67     * 15.4    60    control           77     * 43.5    348   control           80     * 56.9    419   Hydrocephalus     0      * 150 29.35                                              26.78    413   Adenoleukodystrofy                            13     * 23.1    376   Paresthesia       14     * 21.1    239   Epilepsy, encephalitis                            16     * 29.8    7     SSPE              17     * 48.1    139   Cerebellitis (Mycoplasma p.)                            18     * 19.7    180   Herpes encephalitis                            19     * 14.1    206   CBS               20     * 15.9    140   TC ?              21     * 14.2    228   Ishernic cerebral infarct                            22     * 150    197   Kawasaki          24     * 27.8    133   MS                24     * 37.1    212   Alcohol PNP       25     * 14    66    MS ?              25     * 14    143   Myopathy          25     * 18.3    82    MS                26     * 20.2    258   Guillian-Barre    26     * 14.4    105   MS                26     * 18.4    169   Dementia ?        27     * 37.5    351   Viral meningitis  28     * 17.3    213   MS                28     * 15.7    253   Encephalitis viral ?                            28     * 38.1    236   Migraine          29     * 17.3    405   Guillian-Barre    29     * 47.6    72    MS                29     * 14    234   MS                29     * 19.9    128   GBS               29     * 14    138   PNP               32     * 17.6    218   Empty sella       35     * 14    135   MS                35     * 16.3    346   Neuritis optico   35     * 14    117   Trigeminus neurology                            36     * 14    350   MS                37     * 14    384   Guillian-Barre    37     * 14    123   CVA               38     * 14    56    TIA (transient ischemic                            39     * 14.9          attack)    172   CVA ? psych.      40     * 21.7    189   Hemicranial headache                            41     * 15.9    119   Amyloidosis       42     * 15.3    69    Meningitis        42     * 26.5    231   Contuno alcohol   42     * 15.5    130   External oftalmoplegia                            43     * 53.6    357   CVA               44     * 16.6    391   MS                44     * 14    62    MS ??             44       21.2    124   Syphilis          45     * 20.4    89    PNP               45     * 27.1    122   Cauda equina syndroom etiol.                            46     * 14    249   MS                47     * 14    112   MS ?              48     * 14.9    363   Encephalitis      48     * 35.9    125   MS                48     * 24.9    205   GBS               49    388   MS                50     * 14    418   Tetanos           50     * 14    207   GBS ?             50     * 70.3    114   Cerebellar atrofy 51     * 51.5    121   Syphilis          51     * 28.1    35    MS + PNP (diabetic)                            51     * 17.3    215   OLM               52     * 14    101   Brain infarct     53     * 52.8    255   PNP + (MS ?), diabetic                            53     * 18.4    173   Lyme disease      54     * 17.5    360   (Borrelia) (MS-like)                            54     * 34.7    374   ALS               54     * 65.3    179   Lyme disease      54     * 16.4    50    Epilipsy-alcoholism                            55     * 21.5    184   Epilepsia         55     * 16.7    210   MS                56     * 16.2    58    CVA               57     * 25.3    137   Pick ?            57     * 77.4    131   Meningial bleeding                            57     * 66.7    398   Meningoencephalis 58     * 117.8    349   Meningoencephalis 58     * 35.6    244   Facialis parese   58     * 18.5    219   Pseudobullair syndrome                            58     * 39.6    64    MS                58     * 40.7    36    TIA               59     * 14    240   Guillian-Barre    68?    * 14  25.06                                              15.69    70    N. ulnaris/Parkinson (?)                            60     * 23.8    166   tbc               60     * 14    85    Aneurism          60     * 18.3    204   Bipyramidal idiopathy                            60     * 16.8    167   Cerebellar atrophy                            61     * 14    157   Bulbar paralysis  62     * 17.4    30    Steele Richardson 62     * 19.2    153   Lymfora CSZ       62     * 22.2    414   Subacute polyneuropathy                            62     * 14    37    OPCA              63     * 26.7    109   Pick; ALS         63     * 40.5    277   COLD              64     * 14    195   E, dialysis       64     * 14    148   MS                65     * 15.4    375   Polyneuropathy, Charot-                            65     * 15.5          Marie-Tooth    182   ALS               65     * 14    230   Parkinson         66     * 21.1    409   Multiple vascular (pons.)                            66    170   Temp. E           66     * 24.2    186   ALS               66     * 21.2    59    Menigeal aneurysma                            67     * 28.4    233   Korsakow-like post trauma                            67     * 29.6    120   ALS               67     * 25.7    550   PNP, CVA, diabetes                            67     * 64.2    110   Diabetes          67     * 14    259   Polyvascular syndrome +                            68          demyelinisation    248   (?)               68     * 14    235   CVA               68     * 77.9    362   Guillain-Barre    68     * 14    208   PNP-zona          68     * 28.8    115   Subacute combined 68     * 14          degeneration    98    TIA               68     * 34.9    193   GBS               68     * 44.1    222   Guillain-Barre    69     * 14.1    102   PNP               70     * 14    242   Bipyramidal syndrome PNP                            72     * 20.8    251   Trauma cerebri commoti                            77     * 14    422   Infarct           78     * 20.7    42    Multi-infarct dementia                            78     * 38.9    93    Diabetes, PNP, radiculopathy                            85     * 61.9    53    Mixed dementia, Parkinson                            85     * 59.3    ______________________________________     Abbreviations:     SSPE: Subacute sclerosing panencephalitis,     GBS: GuillainBarre syndrome,     TC: Meningeal tuberculosis,     MS: Multiple sclerosis,     PNP: Polyneuropathy,     CVA: cerebrovascular amyloidosis,     ALS: Amyotrophic lateral sclerosis,     TIA: Transient ischemic attack,     OPCA: Olivoponto cerebellar atrophy,     COLD: Chronic obstructive lung disease.

Example V

Definition of the AT120 epitope

Since AT120 reacts equally well with all isoforms of tau (Goedert etal., 1989), the smallest recombinant tau form was used for deletionmapping. To this, two sites were used for deletion construction, theSacII site at position 155 of the human tau 34 sequence and the SmaIsite position 220 (Goedert et al., 1989). A mTNF-fusion vectorpmTNF(MPH) (Innogenetics, Ghent, Belgium), in which the smallest tauopen reading frame was fused to 25 amino acids of mouse tumor necrosisfactor, was cut with ApaI-SacII, blunted with T₄ DNA polymerase andligated. After cutting the ligated material with Sac II and Apa I toreduce non-mutant background, the mixture was transformed into MC1061 pcI587 (Casadaban & Cohen, (1980)). Each selected clone was furthercharacterized by restriction digestion and by its reactivity withanti-tau antibodies.

The same mouse TNF fusion tau vector was used to insert a frame shiftmutation in the XmaI restriction site. The vector was cut with SmaI,blunted with T₄ DNA polymnerase and the ligated mixture was retreatedwith SmaI before transformation in order to reduce non-mutantbackground. The reactivity pattern of each of the mutants was checkedwith AT120 monoclonal antibodies by means of Western Blotting. Thisallowed to localize the epitope of AT120 in a first approximation to theregion of 65 amino acids spanning amino acid 155-221 which isrepresented in SEQ ID NO 1 (FIG.4). However, since in this region alsotwo other antibodies, BT2 (Mercken et al, 1992a) and HT7 (Mercken, Ph.d.Thesis) show reactivity it was mandatory to prove that no competitionbinding was observed between the latter monoclonal antibodies and AT120.

Therefore, a competition ELISA with each of these antibodies wasperformed. To this, affinity purified rabbit anti-human tau polyclonalantibodies were coated overnight at 4° C. in coating buffer (10 mM TrispH 8.6, 10mM NaCl, 10mM NaN₃), and after blocking with 0,1% caseine inPBS, 100 μl/well of pure PHF-tau was added for 1 h at 37° C. Afterwashing, 50 μl of either AT8, BT2 or AT120 unlabeled monoclonal antibodywas added at a concentration of 10μg/ml and incubation was continued for30 min at 37° C. Next, 50 μl of biotinylated monoclonal antibody wasadded. Each of these biotinylated antibodies was used in a presetconcentration, which in a tau Sandwich ELISA (as described in ExampleII) gave 50% of the maximal OD value. After a subsequent incubation of 1h at 37° C., the plates were further treated as described in Example II.

                  TABLE III    ______________________________________    Competition ELISA using monoclonal antibodies AT8, AT120 and BT2.                 Unlabeled competitor                 Absorbance    BIOTINYLATED AT8         AT120   BT2    ______________________________________    AT8          0.001       0.372   N.D.    AT120        0.476       0.001   N.D.    BT2          N.D.        0.543   0.054    ______________________________________     N.D. refers to not determined absorbances

The results, shown in Table III clearly indicate that the epitoperecognized by the monoclonal antibody AT120 is different from theepitopes recognized by the monoclonal antibodies BT2 and AT8.Consequently, solid phase immobilized synthetic nonapeptides, thesequence of which was derived from the epitope of the invention asdepicted in SEQ ID NO 1, were incubated with each of these monoclonalantibodies. The complexes were visualised as in Example I, section 3. Onthe basis of their respective reactivity patterns it was confirmed thatthe epitope recognized by AT120 is different from that of each of theother monclonal antibodies (Table IV and FIG. 5)

                  TABLE IV    ______________________________________    Reactivity of monoclonal antibodies BT2, HT7    and AT120 with solid phase bound nonapeptides.    The nonapeptides are designated in the one-letter amino acid code.               Detecting Monoclonal antibody               Absorbance    Nonapeptides HT7         BT2    AT120    ______________________________________    GAAPPGQKG    3.00        0.168  0.089    (SEQ ID NO 5)    GDRSGYSSP    0.493       3.00   0.553    (SEQ ID NO 6)    ______________________________________

In addition to these experiments, the AT120 epitope was also confirmedby peptide mapping. Hereto, a total of 20 μg of mTNF-MPHtau1 (see FIG.6) was lyophilized and redissolved in 56 μl of 100 mM Tris HClcontaining 10% acetonitrile. To this was added 14 μl of endoprotease AspN (0.04 ug/ml) and the mixture was incubated for 8 hours at 37° C.Following cleavage, the mixture was divided equally over 5 sample wellsand subjected to SDS-polyacrylamide gel electrophoresis. Followingelectrophoresis, the peptides in the gel were blotted onto a PVDFmembrane. A strip of the PVDF membrane corresponding to one lane on thegel was removed. Free binding sites on the membrane were blocked byincubation with 0.5% casein in PBS, and the strip was incubated with themonoclonal antibody AT120 to determine if protein fragments were presentwhich were capable of being recognized. The presence of bound antibodywas detected using an alkaline phosphatase-labeled rabbit anti-mouseconjugate followed by incubation with 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium. The remainder of the PVDF membranewas stained with Amido Black, which revealed the presence of 5 bands,all with a molecular size smaller than that of the original protein. Oneof these bands was visible on the portion of the blot which wasincubated with AT120. The band which was specifically recognized byAT120 corresponded to the largest of the Amido Black-stained bands withan apparent molecular size of approximately 6 kDa. This band was excisedfrom the Amido Black-stained blot and subjected to amino terminalsequence analysis (Edman degradation). The analysis revealed that theband recognized by AT120 began with Asp193 in the amino acid sequence oftau, close to the C-terminus of the tau sequence contained within themTNF-MPHtau1 recombinant protein.

It thus appeared probable, that additional amino acid sequences locateddownstream with respect to Arg221 might be required for binding of themonoclonal antibody AT120. In order to test this hypothesis in anattempt to further delineate the boundaries of the epitope, peptide(tau5B) was synthesized: ##STR5##

Since it cannot be excluded that the second glycine residue of thespacer sequence is functionally equivalent to Gly207, the position ofthe epitope bound by AT120 must lie between Gly207 and Thr231.

In order to localize the epitope within the sequence of peptide tau5Beven more precisely, overlapping 12-mers spanning the tau5B sequencewere synthesized in quadruplicate as defined spots on Whatman 1MM paper.The paper was first derivatized with two residues of β-alanine toprovide the paper with a handle on which peptide synthesis could takeplace. The first of these β-alanine residues was coupled as a symmetricanhydride in the presence of 1 equivalent of dimethylaminopyridine. Thesecond β-alanine residue was coupled following in situ activation using2-(1H-benzotriazol-1-yl)-1,1,3 ,3-tetramethyluronium tetrafluoroboratein the presence of 1-hydroxybenzotriazole. The coupling of alladditional amino acids was performed using the same chemistry (Knorr etal., 1989). The peptides synthesized and their recognition by AT120 wereas follows:

    ______________________________________                             REACTION    PEPTIDE SEQUENCE         WITH AT120    ______________________________________    GGSRSRTPSLPT (SEQ ID NO 8)                             -     GSRSRTPSLPTP (SEQ ID NO 9)                             -     SRSRTPSLPTPP (SEQ ID NO 10)                             -      RSRTPSLPTPPT (SEQ ID NO 11)                             -      SRTPSLPTPPTR (SEQ ID NO 12)                             -       RTPSLPTPPTRE (SEQ ID NO 13)                             -       TPSLPTPPTREP (SEQ ID NO 14)                             -        PSLPTPPTREPK (SEQ ID NO 15)                             +++        SLPTPPTREPKK (SEQ ID NO 16)                             +++         LPTPPTREPKKV (SEQ ID NO 17)                             +++         PTPPTREPKKVA (SEQ ID NO 18)                             +++          TPPTREPKKVAV (SEQ ID NO 19)                             +++          PPTREPKKVAVV (SEQ ID NO 20)                             +++           PTREPKKVAVVR (SEQ ID NO 21)                             -           TREPKKVAVVRT (SEQ ID NO 22)                             -    ______________________________________

From this experiment, it is evident that the epitope recognized by AT120is a linear sequence comprised of amino acids Pro218 to Lys224 asspecified in SEQ ID NO 4.

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    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 24    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 67    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    ArgGlyAlaAlaProProGlyGlnLysGlyGlnAlaAsn    1510    AlaThrArgIleProAlaLysThrProProAlaProLys    152025    ThrProProSerSerGlyGluProProLysSerGlyAsp    3035    ArgSerGlyTyrSerSerProGlySerProGlyThrPro    404550    GlySerArgSerArgThrProSerLeuProThrProPro    556065    ThrArg    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    SerProGlySerProGlyThrProGlySerArgSerArg    1510    ThrProSerLeuProThrProProThrArgGluProLys    152025    LysValAlaValValArgThr    30    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    GlySerArgSerArgThrProSerLeuProThrProPro    1510    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    ProProThrArgGluProLys    15    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    GlyAlaAlaProProGlyGlnLysGly    15    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    GlyAspArgSerGlyTyrSerSerPro    15    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    GlyGlySerArgSerArgThrProSerLeuProThrPro    1510    ProThrArgGluProLysLysValArgValValArgThr    152025    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    GlyGlySerArgSerArgThrProSerLeuProThr    1510    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    GlySerArgSerArgThrProSerLeuProThrPro    1510    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    SerArgSerArgThrProSerLeuProThrProPro    1510    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    ArgSerArgThrProSerLeuProThrProProThr    1510    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    SerArgThrProSerLeuProThrProProThrArg    1510    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    ArgThrProSerLeuProThrProProThrArgGlu    1510    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    ThrProSerLeuProThrProProThrArgGluPro    1510    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    ProSerLeuProThrProProThrArgGluProLys    1510    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    SerLeuProThrProProThrArgGluProLysLys    1510    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    LeuProThrProProThrArgGluProLysLysVal    1510    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    ProThrProProThrArgGluProLysLysValAla    1510    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    ThrProProThrArgGluProLysLysValAlaVal    1510    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    ProProThrArgGluProLysLysValAlaValVal    1510    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    ProThrArgGluProLysLysValAlaValValArg    1510    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    ThrArgGluProLysLysValAlaValValArgThr    1510    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 391    (B) TYPE: Amino Acid    (C) STRANDEDNESS: Unknown    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    MetValArgSerSerSerGlnAsnSerSerAspLysPro    1510    ValAlaHisValValAlaAsnHisGlnValGluGluGln    152025    GlyIleHisHisHisHisHisHisValAspProGlyPro    3035    MetAlaGluProArgGlnGluPheGluValMetGluAsp    404550    HisAlaGlyThrTyrGlyLeuGlyAspArgLysAspGln    556065    GlyGlyTyrThrMetHisGlnAspGlnGluGlyAspThr    7075    AspAlaGlyLeuLysAlaGluGluAlaGlyIleGlyAsp    808590    ThrProSerLeuGluAspGluAlaAlaGlyHisValThr    95100    GlnAlaArgMetValSerLysSerLysAspGlyThrGly    105110115    SerAspAspLysLysAlaLysGlyAlaAspGlyLysThr    120125130    LysIleAlaThrProArgGlyAlaAlaProProGlyGln    135140    LysGlyGlnAlaAsnAlaThrArgIleProAlaLysThr    145150155    ProProAlaProLysThrProProSerSerGlyGluPro    160165    ProLysSerGlyAspArgSerGlyTyrSerSerProGly    170175180    SerProGlyThrProGlySerArgSerArgThrProSer    185190195    LeuProThrProProThrArgGluProLysLysValAla    200205    ValValArgThrProProLysSerProSerSerAlaLys    210215220    SerArgLeuGlnThrAlaProValProMetProAspLeu    225230    LysAsnValLysSerLysIleGlySerThrGluAsnLeu    235240245    LysHisGlnProGlyGlyGlyLysValGlnIleValTyr    250255260    LysProValAspLeuSerLysValThrSerLysCysGly    265270    SerLeuGlyAsnIleHisHisLysProGlyGlyGlyGln    275280285    ValGluValLysSerGluLysLeuAspPheLysAspArg    290295    ValGlnSerLysIleGlySerLeuAspAsnIleThrHis    300305310    ValProGlyGlyGlyAsnLysLysIleGluThrHisLys    315320325    LeuThrPheArgGluAsnAlaLysAlaLysThrAspHis    330335    GlyAlaGluIleValTyrLysSerProValValSerGly    340345350    AspThrSerProArgHisLeuSerAsnValSerSerThr    355360    GlySerIleAspMetValAspSerProGlnLeuAlaThr    365370375    LeuAlaAspGluValSerAlaSerLeuAlaLysGlnGly    380385390    Leu    (2) INFORMATION FOR SEQ ID NO: 24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1173    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Double Stranded    (D) TOPOLOGY: Unknown    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:    ATGGTAAGATCAAGTAGTCAAAATTCGAGTGACAAGCCTG40    TAGCCCACGTCGTAGCAAACCACCAAGTGGAGGAGCAGGG80    AATTCACCATCACCATCACCACGTGGATCCCGGGCCCATG120    GCTGAGCCCCGCCAGGAGTTCGAAGTGATGGAAGATCACG160    CTGGGACGTACGGGTTGGGGGACAGGAAAGATCAGGGGGG200    CTACACCATGCACCAAGACCAAGAGGGTGACACGGACGCT240    GGCCTGAAAGCTGAAGAAGCAGGCATTGGAGACACCCCCA280    GCCTGGAAGACGAAGCTGCTGGTCACGTGACCCAAGCTCG320    CATGGTCAGTAAAAGCAAAGACGGGACTGGAAGCGATGAC360    AAAAAAGCCAAGGGGGCTGATGGTAAAACGAAGATCGCCA400    CACCGCGGGGAGCAGCCCCTCCAGGCCAGAAGGGCCAGGC440    CAACGCCACCAGGATTCCAGCAAAAACCCCGCCCGCTCCA480    AAGACACCACCCAGCTCTGGTGAACCTCCAAAATCAGGGG520    ATCGCAGCGGCTACAGCAGCCCCGGCTCCCCAGGCACTCC560    CGGCAGCCGCTCCCGCACCCCGTCCCTTCCAACCCCACCC600    ACCCGGGAGCCCAAGAAGGTGGCAGTGGTCCGTACTCCAC640    CCAAGTCGCCGTCTTCCGCCAAGAGCCGCCTGCAGACAGC680    CCCCGTGCCCATGCCAGACCTGAAGAATGTCAAGTCCAAG720    ATCGGCTCCACTGAGAACCTGAAGCACCAGCCGGGAGGCG760    GGAAGGTGCAAATAGTCTACAAACCAGTTGACCTGAGCAA800    GGTGACCTCCAAGTGTGGCTCATTAGGCAACATCCATCAT840    AAACCAGGAGGTGGCCAGGTGGAAGTAAAATCTGAGAAGC880    TTGACTTCAAGGACAGAGTCCAGTCGAAGATTGGGTCCCT920    GGACAATATCACCCACGTCCCTGGCGGAGGAAATAAAAAG960    ATTGAAACCCACAAGCTGACCTTCCGCGAGAACGCCAAAG1000    CCAAGACAGACCACGGGGCGGAGATCGTGTACAAGTCGCC1040    AGTGGTGTCTGGGGACACGTCTCCACGGCATCTCAGCAAT1080    GTCTCCTCCACCGGCAGCATCGACATGGTAGACTCGCCCC1120    AGCTCGCCACGCTAGCTGACGAGGTGTCTGCCTCCCTGGC1160    CAAGCAGGGTTTG1173    __________________________________________________________________________

We claim:
 1. A method for detecting a disease involving alterations intau protein by the quantification of total phosphorylated andunphosphorylated tau protein comprising:(a) contacting a sample from apatient suspected of suffering from Alzheimer's disease or any otherdisease involving alterations in tau protein with a monoclonal antibodyproduced by the hybridoma AT120 under conditions that form atau-antibody complex, wherein the sample is selected from the groupconsisting of cerebrospinal fluid, serum, a neurofibrillary tanglepreparation, and a detergent extracted brain homogenate; (b) quantifyingthe amount of total tau by detecting the amount of total phosphorylatedand unphosphorylated tau protein in the tau-antibody complex, wherein anincrease in the amount of total tau when compared to a normal healthycontrol is indicative of disease.
 2. A method for detecting a diseaseinvolving alterations in tau protein by the quantification of totalphosphorylated and unphosphorylated tau protein comprising:(a)contacting a sample isolated from a patient suspected of suffering froma neurological disorder involving alterations in tau protein with amonoclonal antibody produced by the hybridoma AT120 under conditionsthat form a tau-antibody complex; (b) quantifying the amount of totaltau by detecting the amount of total phosphorylated and unphosphorylatedtau protein in the tau-antibody complex, wherein an increase in theamount of total tau when compared to a normal healthy control isindicative of disease.
 3. The method of claim 2 wherein the sample to betested is an unconcentrated cerebrospinal fluid (CSF) sample.
 4. Themethod of claim 2 wherein the disorder is Alzheimer's disease.